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a Department of Obstetrics and Gynecology, CSMC Burns and Allen Research Institute, UCLA School of Medicine,Los Angeles, California 90048-0750
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using mouse brain [8], pancreatic ß cells [9], and transfected cell lines [10], it has been demonstrated that leptin binds with high affinity to a cell-surface receptor (OB-R). OB-R is structurally similar to the class I cytokine receptor gp130 subunit [10]. Three major classes of leptin receptor isoforms have been identified in the rat brain: 1) the long, signal-transducing isoform, OB-Rb; 2) truncated isoforms, OB-Ra, OB-Rc, and OB-Rd; and 3) a soluble isoform, OB-Re [11]. The cellular distribution of leptin receptor isoforms within the rodent ovary has not been reported.
Upon receptor activation, an intricate signaling cascade that is mediated by one or more Janus kinase isoforms, and signal transducers and activators of transcription (STAT) proteins has been shown to mediate leptin bioactivity [10]. At present, the intracellular mechanisms that regulate leptin bioactivity within the ovary are unknown.
In the ovary, FSH is required for the recruitment of small antral follicles into the growing preovulatory cohort. As a consequence of FSH stimulation, the granulosa cells (GC) differentiate into estradiol-17ß (E2)-producing cells, and E2 is required for continued follicle viability. It has become apparent that a group of intraovarian growth factors and cytokines modulates the FSH-dependent growth and differentiation of GC [12]. Many of these regulatory factors have redundant actions in GC. For example, both insulin-like growth factor-I (IGF-I) and transforming growth factor ß (TGF-ß) augment FSH-dependent E2 production. Hence, TGF-ß appears to be a key supportive intraovarian factor because it promotes GC growth [13] and augments FSH-dependent E2 synthesis [14].
Leptin has been shown to exert direct inhibitory effects on ovarian GC. In cultures of rat [5], bovine [6], and human [7] GC, leptin suppressed the sensitizing effect of insulin and/or IGF-I on FSH-dependent E2 production. Because E2 production is essential for ovarian follicle growth and ovulation, and IGF-I appears to be an obligatory mediator of FSH-dependent follicle development [15], it appears that leptin can interfere with an important regulatory mechanism supporting follicle viability and ultimately ovulation. In light of the redundant modulation of FSH action by growth factors and cytokines, it was of interest to determine whether leptin caused a general antagonism of the stimulatory modulators of FSH-dependent E2 production. This study tested the effects of leptin on TGF-ß regulation of E2 production by GC, and investigated intraovarian mechanisms whereby leptin suppresses E2 synthesis in GC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant murine leptin (carrier-free) and recombinant human transforming growth factor ß1 (TGF-ß; lyophilized with BSA as carrier) were purchased from R&D Systems (Minneapolis, MN). Human recombinant FSH and ovine LH were supplied by the National Hormone and Pituitary Program of the NIDDK, NICHD, and USDA (Rockville, MD). McCoy's 5a medium (M5a, serum-free) and Medium 199 were purchased from GIBCO-BRL (Grand Island, NY). Culture plates were purchased from Falcon (Lincoln Park, NJ). [1ß-3H(N)]Androstenedione (A4; 21.5 Ci/mmol) was obtained from Dupont NEN (Boston, MA). The E2 RIA kit was obtained from Diagnostic Products Corporation (Los Angeles, CA). The estrone (E1) RIA kit was obtained from Diagnostic Systems Laboratories (Webster, TX). Unless otherwise specified, all assay reagents were purchased from Sigma (St. Louis, MO).
GC Culture
All procedures using live animals were approved by the CSMC Institutional Animal Care and Use Committee. Immature (26-day-old) Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) were killed via CO2 inhalation followed by cervical dislocation. Ovaries were removed and placed in ice-cold Medium 199 supplemented with 0.1% BSA. Ovaries were cleaned of bursa and other extraneous tissues, and GC were collected from the surrounding medium after follicle puncture [16]. GC were centrifuged (250 x g) and resuspended in a known volume of M5a, supplemented with penicillin (100 U/ml), streptomycin sulfate (100 µg/ml), and L-glutamine (2 mM). GC number and viability were determined by trypan blue exclusion using a hemacytometer.
Aliquots containing 50 000–60 000 viable GC were placed in 96-well culture plates. GC were incubated in a final volume of 200 µl M5a/well containing 0.1 µM A4 at 37°C in a humidified atmosphere containing 5% CO2 in air. Control GC were incubated without additional hormones. Designated GC were challenged with FSH (0.001–1.0 IU/ml) with or without TGF-ß (10 ng/ml). Separate cultures were treated with FSH (0.001–1.0 IU/ml) plus leptin (10 ng/ml) with and without TGF-ß (10 ng/ml). The leptin concentration was chosen on the basis of the reported Kd (0.7 nM) for leptin binding [8] and previous studies by our lab [5], as well as serum leptin concentrations in the human [17]. Cultures were terminated at 48 h, and the conditioned media were collected and frozen at -20°C pending RIAs to measure E2 and E1 content. RIAs were conducted according to the manufacturers' protocols.
Theca-Interstitial Cell (TIC) Culture
To measure the effect of leptin on androgen production, purified populations of TIC were obtained from the enzymatically dispersed ovaries of 26-day-old hypophysectomized rats as previously described [18]. TIC viability was determined using trypan blue exclusion. TIC were incubated in 96-well plates (approximately 4 x 105 viable TIC/well) in a final volume of 200 µl. TIC were either cultured in M5a alone (control), leptin (0.1, 1.0, 10, and 100 ng/ml), or LH (0.03–10 ng/ml) to induce steroidogenic differentiation of the cells. Designated TIC were challenged with LH in the presence of TGF-ß (10 ng/ml), with and without leptin (10 ng/ml). TIC were incubated for 48 h at 37°C in a humidified atmosphere containing 5% CO2 in air. Cultures were terminated at 48 h, and media were removed and frozen at -20°C until analyzed for androsterone content by RIA [19]. The cells were frozen at -80°C pending extraction of RNA.
Leptin Receptor (OB-R) Isoform mRNA Expression
GC and TIC were harvested after the 48-h incubation period described above. For all reverse transcription (RT)-polymerase chain reaction (PCR) reactions, total RNA, DNA, and protein were extracted from the cells using the Tri Reagent method, according to the manufacturer's protocol (Molecular Research Center, Inc., Cincinnati, OH). Four replicate wells were pooled from GC and TIC cultures, and RT was performed as described [20]. Previous studies have shown that the multiple isoforms of the leptin receptor present in rat hypothalamus can be grouped into three classes: short forms with truncated intracellular domains (OB-Ra, OB-Rc, OB-Rd), the full-length signal-transducing isoform (OB-Rb), and a soluble isoform lacking the transmembrane and intracellular domains (OB-Re) [11]. Therefore, as a positive control, RNA was extracted from fresh hypothalamus tissue harvested from intact 26-day-old female Sprague-Dawley rats. All samples were amplified using oligonucleotide primers (synthesized by GIBCO-BRL) previously shown to amplify the leptin receptor isoforms OB-Ra, OB-Rb, and OB-Re [11]. After 35 cycles of PCR (94°C, 1 min; 52°C, 1 min; 72°C, 1.5 min), the amplification products were separated on a 2% agarose gel and visualized with ethidium bromide.
Measurement of Aromatase Cytochrome P450 (P450arom) mRNA
In order to determine the effect of leptin on P450arom mRNA expression, RNA was extracted from GC cultures as described above. P450arom mRNA was measured using semiquantitative RT-PCR. Primers (sense: 5'-ACT GTG CCT GTC AGT GCC AT-3'; antisense: 3'-GAC CAG AAT AAG CTT ACC A-5') were synthesized in our lab (using an Applied Biosystems model 391 DNA synthesizer, Foster City, CA) and were designed to amplify a 426-base pair (bp) segment of the rat P450arom cDNA [21]. To control for variations in individual PCR reactions, a mutant control P450arom cDNA fragment was synthesized by site-directed mutagenesis [22]. In the P450arom cDNA, a C was substituted for a T at base 320 to introduce an MspI restriction site. The resultant mutant cDNA can be amplified by the P450arom primers but can be distinguished from the amplified wild-type P450arom cDNA by digestion with MspI. The control cDNA (1 pg) was included in each PCR reaction (25 cycles: 94°C, 1 min; 55°C, 2 min; 72°C, 1 min), and all samples from each experiment were amplified at the same time in the presence of [32P]dCTP. The amplification products were separated on a 2% agarose gel and visualized with ethidium bromide. The individual bands were cut from the gel and counted in a ß-spectrometer. P450arom mRNA values were normalized to ß-actin mRNA levels measured [23] in the same samples to account for procedural variability and differences in cell number.
P450arom Activity
P450arom activity was estimated by measuring the production of 3H2O from [1ß-3H]-A4 [24]. GC (5 x 105 viable GC/well, 1 ml final volume) were incubated in 6-well plates without hormones (control), with FSH alone (0.1 IU/ml), with FSH plus TGF-ß (10 ng/ml), and with a combination of FSH, TGF-ß, and leptin (10 ng/ml). After 48 h, fresh hormones were added to the appropriate wells and [1ß-3H]-A4 (2 x 106 cpm, 0.1 µM) was added to all wells. After a 4-h incubation at 37°C, the media were removed and the amount of 3H2O produced was measured [24]. Briefly, trichloroacetic acid (TCA) was added, and the precipitated proteins were removed by centrifugation at 1700 x g for 15 min. The supernatants were collected, and 1 ml of H2O-saturated chloroform was added. The reactions were vigorously shaken for 5 sec; then the aqueous phase was aspirated from each tube and mixed with an ice-cold 5% charcoal, 0.5% Dextran T-70 solution to remove the unreacted substrate. The mixtures were centrifuged (1700 x g, 15 min). The supernatants were collected, scintillation fluid was added, and then the supernatants with scintillation fluid added were counted in a ß-spectrometer. To control for variations in cell numbers, GC were scraped from the wells and protein levels were measured using the Bradford method [25].
Statistical Analyses
Treatments were administered in quadruplicate, and each experiment was repeated a minimum of three times. Mean values from independent experiments were statistically analyzed by unpaired t-test, and multiple comparisons were performed using one-way ANOVA followed by Tukey's test. Values were determined to be significant when P 
0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We examined the cell-specific expression of OB-Ra, OB-Rb, and OB-Re in the immature rat ovary. In GC, the mRNA for OB-R isoforms OB-Ra (Fig. 1, lane 1) and OB-Rb (Fig. 1, lane 2) were expressed. In contrast, TIC expressed only the OB-Ra mRNA (Fig. 1, lane 1). OB-Re mRNA was not detected in either GC or TIC (Fig. 1, lane 3).
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We tested the direct effect of leptin on androgen production in TIC. As shown in Figure 2, there was no effect of leptin on LH-dependent androgen production in the presence or absence of TGF-ß. Leptin (0.1–100 ng/ml) did not alter LH-dependent androsterone levels after 48 h in vitro (data not shown). These results showing a lack of an effect of leptin on TIC androgen production are consistent with the observation that the TIC did not express the signal-transducing isoform of the leptin receptor (OB-Rb).
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Effect of Leptin on GC Estrogen Production
FSH stimulated E2 production in GC (Fig. 3). The maximal stimulatory effect of FSH was detected in the presence of 0.1 and 1.0 IU/ml FSH. In the presence of 0.1 and 1.0 IU/ml FSH, TGF-ß augmented FSH-dependent E2 accumulation by 2.7- and 1.45-fold, respectively (Fig. 3). Leptin did not significantly alter basal or FSH-dependent E2 production, whereas leptin did impair the synergistic effect of TGF-ß on FSH-stimulated E2 synthesis (Fig. 3).
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In order to determine whether there was a selective effect of leptin on the conversion of E1 to E2 (17ß-hydroxysteroid dehydrogenase activity), E1 levels in GC-conditioned media were measured. As expected, FSH induced a dose-dependent increase in E1 accumulation, and leptin did not alter the FSH effect (Fig. 4). In the presence of FSH (0.01, 0.1, and 1.0 IU/ml), TGF-ß significantly augmented E1 accumulation. When leptin was added to FSH- and TGF-ß-stimulated cells, E1 levels were diminished at the 2 highest concentrations of FSH tested (0.1 and 1.0 IU/ml) (Fig. 4).
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Effects of Leptin on P450arom mRNA Levels and Activity
In order to understand the mechanism of leptin interference with TGF-ß stimulation of estrogen production, the effect of leptin on P450arom mRNA expression was examined. In the absence of FSH, P450arom mRNA expression was not stimulated above control levels by TGF-ß, leptin, or TGF-ß plus leptin (Fig. 5). FSH (0.01 and 0.1 IU/ml) induced an increase in P450arom mRNA above control levels that was not significantly altered by leptin. TGF-ß augmented the stimulatory effect of FSH (0.1 IU/ml) on P450arom mRNA levels (Fig. 5). When GC were treated with leptin in the presence of FSH plus TGF-ß, P450arom mRNA levels were reduced to levels equivalent to FSH-stimulated levels.
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We next measured the effect of leptin on P450arom activity. As shown in Figure 6, P450arom activity was increased in FSH-treated GC compared to untreated control cells. In the presence of TGF-ß, FSH-stimulated P450arom was augmented 4-fold. Addition of leptin reduced the up-regulation in FSH-dependent P450arom activity by 27% (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence support the conclusion that TGF-ß is an important intraovarian regulator that potentiates FSH action in GC. First, TGF-ß mRNAs are expressed by GC [29] and TIC [30], and TGF-ß is secreted by TIC [31]. Second, in rat GC, TGF-ß augments the FSH-stimulated cAMP-dependent second messenger pathway by increasing FSH-induced cAMP levels [32] and synthesis of both cAMP-dependent protein kinase RIIß subunit mRNA and protein [33]. Third, TGF-ß stimulates GC growth [13] and steroidogenesis [14] in vitro. The present study expands this body of knowledge by demonstrating an up-regulatory effect of TGF-ß on FSH-stimulated P450arom mRNA expression and P450arom activity in rat GC.
In previous reports by our laboratory and others, leptin was shown to reduce the synergistic effect of IGF-I and/or insulin on FSH-dependent E2 synthesis in rat [5], bovine [6], and human [7] GC in vitro. These observations raised the question whether the direct effect of leptin in GC was specific to IGF-I or if leptin exerted a generalized effect on multiple positive modulators of FSH action. This report demonstrated that leptin blocks the positive modulatory effects of TGF-ß on FSH-dependent estrogen (E1 and E2) production. Hence, the actions of TGF-ß and IGF-I, two well-characterized stimulatory modulators of GC function, are impaired by leptin. These data support the concept that the intracellular signaling pathways mediating TGF-ß and IGF-I enhancement of P450arom gene transcription in GC converge at a common point. Leptin appears to block P450arom gene transcription distal to the point of convergence.
In the present study, leptin blocked the TGF-ß-dependent increase in FSH-stimulated estrogen synthesis. It appears that there was a greater effect of leptin on estrogen production than on aromatase activity. Whether or not the apparent difference is important is unclear. The concentrations of estrogens measured in conditioned medium reflect the net metabolism of A4 over a 48-h period of time by a combination of steroidogenic enzymes. E2 synthesis from A4 requires the aromatase and 17ß-hydroxysteroid dehydrogenase enzymes and may be influenced by other enzymes such as 5
-reductase that can metabolize the A4 substrate or others that could metabolize the estrogen products. Unlike RIA, the aromatase enzyme assay specifically measures aromatase activity. The results of these experiments indicate that leptin treatment may affect not only aromatase activity but also the activities of other steroidogenic enzymes in granulosa cells.
Leptin mRNA has been detected in human GC [34], and immunoreactive leptin has been found in human follicular fluid [7, 27]. Together the aforementioned show a potential intraovarian leptin system, replete with ligand and one or more OB-R isoforms. Importantly, leptin production by human GC has yet to be demonstrated, and there is no difference between circulating and follicular fluid concentrations of leptin [7, 27], indicating that intraovarian leptin is likely to be of endocrine origin. It appears that the physiologic role of leptin in the ovary may be limited to conditions of obesity. For example, the circulating concentrations of leptin in lean women are too low to alter ovarian function significantly [7]. In contrast, the levels observed in obese women are sufficient to interfere with the sensitizing actions of IGF-I and TGF-ß on FSH-dependent E2 production. Such an effect could inhibit fertility because sensitization of small antral follicles to FSH by intraovarian factors (i.e., IGF-I and TGF-ß) is thought to be important for selection of dominant follicles [35]. Disruption of E2 production during follicle growth could cause follicle atresia. Thus, by counteracting the effect of TGF-ß in GC, leptin may interfere with an essential support mechanism (e.g., augmentation of E2 production) that promotes follicle growth and maturation. Such a mechanism can help to explain how weight loss in obese women can improve their fertility [36].
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FOOTNOTES |
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2 Correspondence: Denis Magoffin, Cedars-Sinai Medical Center, 8700 Beverly Blvd., Davis 2066, Los Angeles, CA 90048-0750. FAX: 310 652 8010; magoffin{at}cshs.org
3 Current address: Southern Illinois University, School of Dental Medicine, Department of Applied Dental Medicine, 2800 College Ave., Alton, IL 62002-4700.
Accepted: June 2, 1999.
Received: January 20, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-hydroxylase messenger ribonucleic acid expression by luteinizing hormone and insulin-like growth factor-I. Endocrinology 1993; 132:1945–1951.This article has been cited by other articles:
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